期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 9, 页码 6664-6672出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.9.6664
关键词
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资金
- NEI NIH HHS [F31 EY006901-03, F31 EY006901, EY06901, EY02687, EY50673, P30 EY002687] Funding Source: Medline
- NIDDK NIH HHS [P60 DK020579, P30 DK020579] Funding Source: Medline
alpha-Crystallin, a large lenticular protein complex made up of two related subunits (alpha A- and alpha B-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract, To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alpha A- and alpha B-crystallins conjugated to a small fluorescent tag (Alexa350(R)). Both alpha A and alpha B homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 +/- 0.11 ng/mu g of membrane and 4.57 +/- 0.50 x 10(-4) mu g(-1) of membrane, respectively, The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.
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